We have characterized HeLa cell microtubules obtained after four cycles of polymerization-depolymerization and have found the major accessory protein (MAP) associated with these microtubules has a MW of 201-206K daltons by SDS-PAGE. This protein is heat-stable and can be increased in amount in HeLa cell extracts by boiling in a water bath. Further, by in vitro reconstitution experiments, this MAP promotes assembly of HeLa cell tubulin to form microtubules at least as efficiently as calf brain MAP-2. Current experiments are aimed at determining if HeLa MAP's are essential for adenovirus edge-binding in vitro to microtubules. On another front we have examined the structural and biochemical alterations in adenovirus particles infecting HeLa cells. Shortly after penetration into cells, the viral density was found to shift to a lower value as revealed by CsCl equilibrium centrifugation. The structural resemblance between penetrating virus and pentonless particles produced in vitro was particularly noted. Further, penetrating particles infecting HeLa cells which had been pregrown in a medium containing 32P-orthophosphate were also found to become phosphorylated at protein IIIa at early times (20-30 min) after infection. The in vivo phosphorylated IIIa protein and IIIa phosphorylated in vitro by viral endogenous protein kinases or by cellular enzymes were compared by both tryptic peptide analysis and phosphoamino acid composition. Considerable similarities were noted. The results suggest that the phosphorylation of IIIa is a phenomenon which essentially takes place during an early stage of infection and may play an important role in viral entry or intracellular transport.